Risk of fatal hypoglycemia due to insulin or its secretagogues is a major limiting factor for optimal glucose control in management of diabetes. Hypoglycemia causes about 6 to 10% of deaths in diabetes patients and costs nearly $1.4 billion annually in the US. Moreover, repeated hypoglycemia leads to hypoglycemia associated autonomic failure (HAAF), which is a phenomenon of progressive decline in counterregulatory response to subsequent episodes of hypoglycemia. To effectively prevent or counteract HAAF, it is imperative that we first understand the mechanisms underlying this potentially fatal condition. We have observed that melanocortin 4 receptor (MC4R) deficiency in the paraventricular nucleus of the hypothalamus (PVH) impairs the stimulation of the release of counterregulatory hormones such as glucagon and epinephrine in response to hypoglycemia in mice (findings from my K01-funded project). Moreover, administration of MC4R agonist in the PVH of diabetic mice restored their counterregulatory response to hypoglycemia. Collectively, these data indicate that normal MC4R signaling is critical to counteract hypoglycemia. Therefore, weakening of the MC4R function can lead to defective hypoglycemia counterregulation. Based on our preliminary data and the role of MC4R in stimulating the sympathetic nervous system activity, it is likely that recurrent hypoglycemia causes autonomic failure by diminishing the function of MC4R in the PVH. Hence, we hypothesize that HAAF is a consequence of dysfunctional MC4R in the PVH and that enhancement of the MC4R function will prevent HAAF in health and diabetes. To test our hypothesis, in Aim 1, we will determine if recurrent hypoglycemia impairs the stimulation of release of counterregulatory hormones by reducing the function of MC4R, which in turn decreases adrenal sympathetic nerve activity to mediate HAAF. Moreover, in Aim 2, we will evaluate the efficacy of genetic and pharmacological enhancement of MC4R function in the PVH to prevent HAAF in health and diabetes. Through my career development award - K01, I have acquired necessary skills and experience to accomplish the goals of this project. Moreover, the University of Rochester has outstanding resources and facilities to support the project. In summary, we will establish whether or not recurrent hypoglycemia diminishes the function of MC4R in the PVH to mediate HAAF. Enhancing MC4R function may emerge as a strategy to prevent HAAF in diabetes. Importantly, this R03 funding program will augment the progress of my K award and facilitate the generation of sufficient preliminary data for my subsequent R01 grant application focusing on further unraveling the MC4R downstream pathways that are responsible for stimulating the counterregulatory response to hypoglycemia and mitigating HAAF.